**Fecal Microbiota Transplantation: Screening and Selection to Choose the Optimal Donor**

**Stefano Bibbò 1,2, Carlo Romano Settanni 1,2, Serena Porcari 1,2, Enrico Bocchino 2, Gianluca Ianiro 1,2, Giovanni Cammarota 1,2 and Antonio Gasbarrini 1,2,\***


Received: 28 April 2020; Accepted: 2 June 2020; Published: 5 June 2020

**Abstract:** In the past decade, fecal microbiota transplantation (FMT) has rapidly spread worldwide in clinical practice as a highly effective treatment option against recurrent *Clostridioides di*ffi*cile* infection. Moreover, new evidence also supports a role for FMT in other conditions, such as inflammatory bowel disease, functional gastrointestinal disorders, or metabolic disorders. Recently, some studies have identified specific microbial characteristics associated with clinical improvement after FMT, in different disorders, paving the way for a microbiota-based precision medicine approach. Moreover, donor screening has become increasingly more complex over years, along with standardization of FMT and the increasing number of stool banks. In this narrative review, we discuss most recent evidence on the screening and selection of the stool donor, with reference to recent studies that have identified specific microbiological features for clinical conditions such as *Clostridioides di*ffi*cile* infection, irritable bowel syndrome, inflammatory bowel disease, and metabolic disorders.

**Keywords:** gu<sup>t</sup> microbiota; precision medicine; *Clostridium di*ffi*cile*; inflammatory bowel disease; ulcerative colitis; irritable bowel disease; metabolic syndrome

#### **1. Fecal Microbiota Transplantation: A New Old Therapy**

In the last decade, multiple studies have expanded knowledge in the field of gu<sup>t</sup> microbiota, including pathogenesis, diagnosis, and therapeutics [1]. To date, the therapeutic modulation of the intestinal microbiota is performed with traditional approaches such as antibiotics and probiotics, or increasingly through fecal microbiota transplantation (FMT), which is defined as the transfer of fecal material from a healthy donor into the gastrointestinal tract of a recipient [2].

Fecal material has been used in medicine since almost two thousand years. The first description of the use of fecal material for medical purposes dates back to about 1700 years ago; traditional Chinese medicine in particular had perceived the potential role of this biological material and used it for several clinical indications such as gastrointestinal, nervous system, skin, and gynecological diseases [3]. In Western countries, the first description of ancestral FMT dates to the 17th century, when Fabricius Acquapendente reported the transplantation of feces for the cure of animals unable to ruminate [4]. More recently, anecdotal use has been reported during the Second World War. German soldiers residing in North Africa suffered from recurrent episodes of diarrhea that they treated by eating camel stool, being inspired from the local practice of the Bedouins [5]. Western medicine began to study the potential role of FMT only in the second half of the 20th century. Firstly in 1958, Ben Eiseman reported the successfully treatment of four patients with pseudomembranous colitis using fecal enemas [6], and over 20 year later, Schwan et al. reported new evidence supporting the e fficacy of FMT in *C. di*ffi*cile* infection (CDI) [7]. In the following years, several other reports came out, and a growing body of evidence showed the e fficacy of FMT in the treatment of recurrent CDI, and, furthermore, the feasibility of FMT was gradually suggested for other clinical indications.

The first randomized controlled trial that investigated the role of FMT for recurrent CDI was published by van Nood et al. in 2013. They reported that a single infusion of fecal material by nasoduodenal route was superior to standard therapy with vancomycin [8]. In further years, other routes of administration were successfully tested in clinical trials, demonstrating the e fficacy of FMT by lower route through colonoscopy [9] or upper administration with capsule [10]. Therefore, the growing interest of the scientific community towards FMT has meant that a large amount of data has been published in the last decade; for this reason, a panel of European experts met in Rome in 2017 to release the first evidence-based consensus report for the use of FMT in clinical practice [11].

Over the years, further issues have emerged, which are still not clarified to date. In particular, in view of the growing number of patients who could benefit from FMT, it is necessary to identify innovative ways to storing fecal material to be used if necessary. Indeed, in the early experiences, FMT was performed only with fresh material from occasional healthy donors, but this approach is not feasible for large-scale use of FMT. To solve this problem, the possibility to create structures to bank the feces after manipulation was suggested, and this approach is supported by the evidence of the e ffectiveness of FMT performed with frozen material [12]. In consideration of the increasing interest of the scientific community on this topic, a panel of international experts met in Rome in 2019 to define the general guidelines for the creation of stool banks [13]. Despite these e fforts, many problems remain to be solved. Above all, the identification of the optimal donor is a fundamental clinical issue of rising relevance. Indeed, the increasing number of clinical indications suggests the need to identify the ideal donor for each disease or patient that cannot be treated indiscriminately with the same fecal biomass. The fascinating idea of identifying the "perfect" intestinal microbiota has motivated the scientific community for at least one century—ever since in the early 20 century Elie Metchniko ff suggested the role of intestinal bacteria in the development of many pathological conditions and health in the homeostasis of the microbial species [14], generating the concepts of "eubiosis" and "dysbiosis," which for years were considered only fascinating hypotheses without strong scientific bases. However, in recent years, the molecular techniques of genomic sequencing have allowed to understand the link between gu<sup>t</sup> microbiota and several diseases [15], giving evidence to this old intuition. In particular, we refer to "eubiosis" as considering a status characterized by a preponderance of potentially beneficial species, while "dysbiosis" is a condition characterized by the loss of homeostasis and by the proliferation of microbial species considered potentially pathogenic and, moreover, favor a "milieu" triggering the hyper-inflammatory state [16]. To date, an increasing number of studies confirm these hypotheses, in particular the reduced diversity of gu<sup>t</sup> microbiota, simply defined as the variety and abundance of species in a defined microbial ecosystem [17,18], which is known to characterize several chronic diseases compared to a control group [19,20]. Therefore, in this narrative review, we report the most recent evidences on the screening and selection of the stool donor, with special e fforts to describe findings that may lead to the optimal donor in several disease looking for an "optimal microbiota" to be transplanted (CDI, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and other emerging pathological conditions).

#### **2. Fecal Microbiota Transplantation in Clinical Practice**

To date, the only recommendation for FMT in clinical practice is the treatment of recurrent CDI, although a large number of emerging indications are being experienced in several studies [21].

CDI is a burdensome clinical issue and represent the most relevant cause of antibiotic-associated diarrhea; its incidence has evolved in recent years and the risk of recurrence after standard antibiotic therapy has widely increased [22,23]. The standard treatment for the first occurrence of CDI is still represented by antibiotic therapy, mainly with metronidazole or vancomycin [24]. However, the clinical success rate of antibiotics in the recurrence of CDI is dramatically decreased, consequently, more effective therapies have been proposed, including FMT [25,26]. The clinical success of FMT, in contrast to the loss of efficacy of standard antibiotic therapy, could be explained by understanding the mechanism of action. In fact, FMT is a restorative treatment of gu<sup>t</sup> microbiota alterations, unlike antibiotics, which is a disruptive treatment; accordingly, the administration of FMT results in a prompt and sustained normalization of microbial community structure and then metabolic activity of gu<sup>t</sup> microbiota [27]. Indeed, CDI develops only in subjects with disruption of gu<sup>t</sup> microbiota [28]; supporting this idea, it was demonstrated that the feces of patients with recurrent CDI have a higher relative abundance of several bacterial family as *Enterobacteriaceae*, *Veillonellaceae*, and *Lactobacillaceae*, and lower relative abundance of *Ruminococcaceae*, *Bacteroidaceae*, and *Lachnospiraceae* [29]. Furthermore, FMT recipients have shown changes in microbial profiles and shifts in the gu<sup>t</sup> microbiota composition towards a profile similar to that of the healthy donor; this finding is obtained in a few days and is observed for at least six months [30].

To date, several systematic review and meta-analyses have shown an overall cure rate of FMT of up to 90% in preventing further CDI recurrence [31,32]. Moreover, a recent meta-analysis has shown that both the upper and the lower route are effective, with a slight advantage of colonoscopy over other techniques [32]. Based on these positive evidences, scientific societies have included FMT among the recommended treatment for recurrent CDI. Already in 2014, FMT was strongly recommended in recurrent CDI by the European Society of Clinical Microbiology and Infectious Disease (ESCMID) [33], while the American College of Gastroenterology (ACG) stated that FMT can be considered after the third recurrence [34]; more recently, the Infectious Disease Society of America (IDSA) confirmed the indication in the treatment of recurrent CDI with FMT [24].

Alongside well-established indications such as CDI, several studies have found emerging clinical conditions for which FMT may represent a promising alternative to standard therapies. Most evidence comes from inflammatory bowel disease (IBD) studies. Several alterations of gu<sup>t</sup> microbiota has been proposed as factors contributing to the development of the aberrant immunological response in IBD [35], but it is still unclear if the perturbations of microbiota are the cause or consequence of the mucosal inflammation associated to IBD [36]. In particular, ulcerative colitis (UC) is the most suitable IBD model for the study of FMT, considering the characteristics of inflammation of the mucosa and the established role of the microbiota in pathogenesis [37]. To date, a little number of clinical trials have reported promising results, but several concerns sugges<sup>t</sup> to better investigate this potential clinical application [38]. According to a Cochrane systematic review of four clinical trials, the overall remission rate at week 8 was 37% (52/140 UC patients) in patients receiving FMT, compared with 18% (24/137 patients) in those receiving placebo; additionally, clinical response and endoscopic remission improved in patients treated with FMT [39]. However, several factors appear to influence the clinical response in UC patients, as the condition during the manipulation of the feces or the donor selection. For instance, anaerobic conditions during the manipulation of stool were associated with better performance considering clinical remission or steroid free response [40]. Donor selection might be a relevant factor considering that a study reported higher success rates with one particular donor compared with other donors [41]. Furthermore, an emerging relevant indication for FMT was represented by the flare of UC associated with concurrent *C. di*ffi*cile* over infection. A recent clinical trial, including patients affected by UC or Crohn disease with recurrent CDI, reported that FMT has a curative effect on the recurrence of CDI, but has no apparent beneficial effect on the IBD course [42].

Gut microbiota disturbance was also involved in other gastrointestinal diseases such as irritable bowel syndrome (IBS). A systematic reviews with meta-analysis showed that FMT may be beneficial in IBS [43], but this finding is limited by the small number of patients included and by the relevant differences in the design of the studies. In particular, IBS is triggered by multiple factors, and furthermore, is a heterogeneous condition that may require a selection of the donor in each case. For instance, El-Salhy et al. have recently reported that FMT administered through gastroscope was

highly e ffective in IBS if a well-defined donor was chosen with a normal disbyosis index and favorable specific microbial signature [44].

Furthermore, metabolic and hepatic diseases are also considered emerging indications for FMT. There is grea<sup>t</sup> interest towards the modulation of the gu<sup>t</sup> microbiota in metabolic syndrome, as two studies reported promising results in improving peripheral insulin sensitivity [45,46]. Unfortunately, the improvement of metabolic profile was not maintained in the long term, and a recent systematic review including three studies reported the absence of significant benefits from FMT in metabolic syndrome [47]. Thus, further studies to clarify the feasibility of this approach in metabolic disorders are needed. Furthermore, FMT was able to reverse encephalopathy derived from disturbed gut-brain axis in patients with liver chronic disease, two clinical studies shown promising results in this field of application [48,49].

FMT was also proposed in the treatment of several other clinical conditions, but evidence is limited and results were reported by small studies; thus, the application is limited to clinical studies and selected cases. For instance, FMT was reported as e ffective in the decolonization of patients carrier of multi-drug resistant organism [50], in reducing symptoms in autism spectrum disorders [51], or in reliving symptoms and increasing progression free survival in graft versus host disease after hematopoietic stem cell transplant [52].

#### **3. Selection and Screening of Stool Donors**

Donor selection represents a fundamental challenge in view of the implementation of FMT programs worldwide. To date, there is a broad debate regarding the preference of donor selection, whether the stool donor should be known to the patient or whether it is preferable to use feces from unrelated donor. Moreover, in the case of non-related donor, fecal material could be banked at dedicated structures that provide support to the hospital that will perform FMT [53].

In particular, the ideal stool donor should be a healthy volunteer, without risk factors for infectious or other chronic diseases, and who is willing to "donate" frequently if needed. Unfortunately, although the conditions do not seem too selective, it is not always easy to identify an adequate number of donors to meet the needs of the FMT program. Indeed, data from large stool bank sugges<sup>t</sup> high rates of donor drop out due to high commitment required [54]; furthermore, physicians often give up FMT because of the complexity and costs of screening [55]. Consequently, to solve these problems, it would be appropriate to implement the undirected donor selection program. Hence, the related donors should be only limited in cases of patient preference. Indeed, undirected donors reduced the likelihood of confidentiality concerns, and then, they are essential for the implementation of stool banking in consideration of easy availability, traceability, and reduction of screening expenses [56].

The screening of potential donors consist in two key landmarks, the preliminary interview and the laboratory testing [13]. A preliminary interview is usually performed by a structured questionnaire that investigated several risk factors to minimize the risk of transferring infections or adverse gu<sup>t</sup> microbiota profile. In particular, the medical interview screen potential donors inquiring about the use of drugs that can alter gu<sup>t</sup> microbiota, known history or risk behaviors for infectious disease, and for disorders potentially associated with the disruption of gu<sup>t</sup> microbiota. The schedule of questions reported in this review (Table 1) includes the most frequently investigated features in leading FMT centers. Obviously, this draft of interview is not mandatory, but can be adapted to the socio-cultural context of potential donors. For example, it would be advisable to carefully investigate the eating habits of potential donors from country where the consumption of raw meat and fish is widespread, thereby increasing the risk of transmission of enteric pathogens, or who eat exotic animals that are potential carriers of unknown pathogens; or seasonal habits that increase the risk to ge<sup>t</sup> infected with intestinal pathogens (e.g., summer holidays and risk of sea food of poor quality). These examples allow to understand how the aim of the interview is to early intercept potential risks of pathogen transmission; thus, each center should adapt the medical interview to its socio-cultural context to make it more e fficient.



The optimal donor correspond at young individual (preferably < 50 years, as suggested by a panel of experts [13] taking into account that increasing age has been associated with altered gu<sup>t</sup> microbiota composition [57]; moreover, aged microbiota could have a negative e ffect contributing to the inflammatory state of the recipient [58]), although is important to exclude candidates with personal history of malignancies or autoimmune disease [13]. Moreover, there are concerns regarding the exclusion of healthcare workers considering the supposed increased risk of colonization by antibiotic-resistant bacteria; however, available data sugges<sup>t</sup> a low prevalence in this population [59].

Potential donors who have a permissive medical history must undergo to blood and fecal examination to exclude infective disease transmittable trough fecal transfer [13]. The tests may change between the various protocols, but there are some mandatory examinations (Table 2).

#### **Table 2.** Donor blood and stool testing.
